1. Field of the Invention
The present invention relates to a novel process for detecting DNA hybridization through impedance measurement using a novel conductive polymer in the DNA biosensor and electrochemical biosensor fields and more particularly to a novel process for detecting DNA hybridization by preparing a novel 3′-carboxyl-5,2′;5′,2″-terthiophene compound; producing a functionalized conductive terthiophene polymer through an electrochemical polymerization using the compound as a monomer on a glassy carbon electrode; immobilizing a probe DNA (or oligonucleotide) onto the polymer and measuring the impedance before and after a hybridization with a target DNA (or oligonucleotide).
2. Related Prior Art
Recently, a need has developed for apparatuses capable of continuously and selectively detecting biological molecules in the biotechnology and medical diagnostic fields. In particular, special attention has been paid to the development of electrochemical biodetectors or biosensors based on DNA hybridization, interactions among drugs and carcinogenic substances and DNAs, and special DNA interactions including DNA damage in the electrochemical detection of nucleic acid. Among these sensors, a variety of DNA sensors have been used to detect DNA sequences, toxic compounds and trace organic compounds. In particular, a hybridization detection method for DNA sequences is recognized as one of most reliable methods in terms of its broad applicability to the genome.
In connection with DNA hybridization detection, some conventional methods for detecting DNA hybridization are known. For example, a method for optically measuring the intensity of fluorescence after hybridizing an immobilized probe DNA with a fluorescent substance(dye)-labeled target DNA (or oligonucleotide), is reported [see, Physicochemical and Engineering Aspects, 2000, 175, 147–152; Analytica Acta, 1997, 350, 51–58; Anal. Chem. 1994, 66, 3379–3383].
A method for measuring the redox of an indicator to detect DNA hybridization is also reported [see, Anal. Chem. 1996, 68, 2629–2634; Anal. Chem. 1994, 66, 3830–3833; Anal. Chem. 2000, 72, 1334–1341]. The method comprises hybridizing an immobilized probe DNA with a target DNA and reacting the hybridized DNAs with the indicator so that the indicator is intercalated into the double-stranded DNA.
Further, disclosed is a method for measuring frequency change using a quartz crystal microbalance (QCM) when a probe DNA immobilized onto the surface of electrode is reacted with a target DNA [see, Biointerfaces, 1998, 10, 199–204; J. Am. Chem. Soc., 1992, 114, 8299–8300].
Further, a method for measuring a potential shift and current change in the redox wave of a conductive polymer (polypyrrole probe) when a probe DNA immobilized onto the conductive polymer is reacted with a target DNA, is disclosed [see, J. Am. Chem. Soc., 1997, 119, 7388–7389; Synthetic Metals, 1999, 100, 89–94].
However, there is a disadvantage in that when a target DNA is labeled with an indicator, the sample preparation is complicated. In addition, when the indicator is inserted after hybridization, time required for experimental steps become long and thus industrially disadvantageous.
In the case of the above-mentioned optical method or the method using QCM, since the measuring instruments used in these methods are huge and expensive, it is necessary for experienced personnel to manage them, as well as the fact that they are unsuitable for portable equipment. In the electrochemical method using a redox indicator, since the degree of hybridization depends on the sensitivity of the indicator, the method's sensitivity and selectivity are problematic.
Further, in the case of the method measuring a redox wave of a polymer, the method is inapplicable to portable sensors.
Therefore, there is a need for a portable DNA sensor which can directly detect DNA hybridization without the use of an indicator thereby shortening the time required in the experiment and which even an inexperienced person can easily identify within a short time whether or not DNA hybridization has occurred.
Thus, the present inventors have earnestly and widely researched the above-mentioned problems in the conventional DNA hybridization detection and have found that when measuring impedance before and after hybridization using a functionalized conductive terthiophene polymer with a novel terthiophene-3-carboxylic acid compound as a monomer, DNA hybridization can be directly detected and as a result they accomplished the present invention.